The present invention relates to a pharmaceutical composition containing 24,25-dihydroxycholecalciferol (hereinafter referred to as 24,25-(OH).sub.2 -D.sub.3 or the present compound) as an active ingredient.
The present inventors have found that 24,25-(OH).sub.2 -D.sub.3 has anti-hypercalcemic activity, anti-ulcer activity, activity of preventing reduction of immunity, activity of controlling magnesium-metabolism, anti-hyperphosphatemic activity, activity of controlling blood sugar level and anti-tumour activity.
It is an object of the present invention to provide a pharmaceutical composition in dosage unit form, comprising a therapeutically effective amount of 24,25-(OH).sub.2 -D.sub.3 and a pharmaceutically acceptable carrier. Another object is to provide a method for treating hypercalcemia, ulcer, reduced immune function, metabolic abnormality on magnesium, hyperphosphatemia, abnormality on blood sugar such as hyperglycemia or tumour, which comprises administering a therapeutically effective amount of 24,25-(OH).sub.2 -D.sub.3 to a human or an animal suffering from the above-mentioned disease. Further object is to provide a process for preparing the pharmaceutical composition. Still other objects will appear hereinafter.
The pharmaceutical activities of the present compound will be described in detail as follows:
Hypercalcemia is a disease of a person whose calcium level in serum has been abnormally raised, and frequently occurs in complication with a disease such as malignant tumour, intoxication due to Vitamin D, sarcoidosis and hyper-parathyroidism.
As a pharmaceutical for treating hypercalcemia, calcintonin has been administered, however, calcitonin which is a peptide hormones is not persistent within the patient's body to which it has been administered and accordingly, there is a problem of durability of its effect. In addition, since calcitonin cannot be orally administered, it must be parenterally administered, for instance, by intramuscular injection, and accordingly, calcitonin has a demerit of being accompanied by the patient's pain. Furthermore, it is pointed out that the administration of calcitonin of non-human origin is accompanied by the reduced effect and anaphylactic shock due to the production of the antibody.
In these situations, the development of an orally administable anti-hypercalcemic agent which is excellently long-lasting in its pharmacological effect has been eagerly awaited.
Concerning the effect of 24,25-(OH).sub.2 -D.sub.3 on calcium in serum, the following reports have been known:
(a) Norman, A. W., J. M. Canterbury, Journal Clin. Invest., 78, 1375-1383, 1978:
It is reported that on the continuous injection of 24,25-(OH).sub.2 -D.sub.3 for 20 min into the healthy dog's thyroid artery, the level of calcium in the dog's serum showed a temporary reduction, however, the level returned to the normal value immediately thereafter. For reference, different from the ordinary administration such as oral-, subcutaneous-, intraperitoneal- and intravenous administration, the continuous injection of an agent into the thyroid artery is a specific method of administration.
(b) Canterbury, J. M., Clin. Res., 26, N1, PA 31, 1978:
It is reported that the administration of 24,25-(OH).sub.2 -D.sub.3 to a dog suffering from hyper-parathyreosis by the thyroid arteral route did not change the dog's calcium level in serum.
(c) Norman, A. W., Brit. Med. J., 280 (No. 6212), 449-450, 1980:
It is reported that the oral administration of 250 micrograms of 24,25-(OH).sub.2 -D.sub.3 to a normal person did not cause the significant change of calcium level in his serum.
(d) Although the following reports concern the administration of 24,25-(OH).sub.2 -D.sub.3 to the patients suffering from hypocalcemia, non of them reported the case where the calcium level in the patient's serum showed a significant change by the administration:
Kanis, J. A., Brit. Med. J., (1978) 1382-1386;
Rasmussen, H., J. Clin. Endocrin. Metab., 46, 284-294 (1978);
Szymendera, J., Brit. Med. J. (Nov. 28) 1465-1466 (1978); and
Kanis, J. A., V-D Basic Research and Its Clinical Appl., (1979), 119-122.
As mentioned above, so far as the present inventors are informed of, there are no reported case where 24,25-(OH).sub.2 -D.sub.3 was administered to human patient of hypercalcemia or animals in the similar morbid state.
As a result of studying and examining the functions of substances present in human body, of which the safety has been confirmed, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 are effective in treating hypercalcemia, particularly in view of its long-lasting pharmacological effect and its possibility of being orally administered, and have arrived at the present invention.
Anti-ulcer activity of the present compound is described as follows:
Peptic ulcer is the ulcer formed on the weakened part of the mucous membrane of stomach or intestine as the damage of substance of the membraneous layer by the action of attacking factor such as hydrochloric acid and pepsin. Although the slight cases are curable by treatment with hospitalization for 3 to 4 months, in the severe cases, hemorrhage and perforation frequently occur and the morbid state becomes chronic.
As the cause of peptic ulcer, the autonomic imbalance and the abnormality of mucous blood flow by the physical and mental stress have been considered, however, since the internal organs themselves are under complicated control of neurohormone, it is practically impossible to elucidate simply to cause of peptic ulcer.
As the conventional anti-ulcer agent, sodium hydrogen carbonate, aluminum salts and magnesium salts have been used for a long time in the sense of neutralizing the acid as one of the attacking factors to the mucous membrane, however, these agents neutralize the acid only temporally to relieve the pain and have scarcely the healing effect on the ulcer.
Recently, anti-ulcer agents based on the presumed cause of ulcerous diseases such as the depressant of autonomic nerve system (so-called anti-choline agent), the reparative agent and the blood flow-improving agent have been numerously developed. However, they are not necessarily satisfactory from the viewpoints of effectiveness or side effects.
Since it takes a long period of time to carry out the treatment of the peptic ulcer, the administration of an anti-ulcer agent frequently extends over a long period of 100 to 150 days on the average, and accordingly, the high treating effect and particularly, the high safety are required to antipeptic ulcer agent.
The present inventors while taking the above-mentioned situation into account have examined the compounds which are endogenously present in a healthy human body, excellent in anti-peptic ulcer activity and pharmacologically quite safe, and as a result of the examination, they have found that 24,25-(OH).sub.2 -D.sub.3 is suitable for their purpose. Our present invention has been completed upon the finding.
The following description relates to the activity of the present compound in preventing the reduction of immunofunction:
Renal failure or uremia is one of the representative diseases which are progressive and bring about extensive reduction of immunity. According to the result of investigation in Japan, 38.3% of infectious cases has been recognized in acute renal failure and 19.2% of infectious cases has been recognized in chronic renal failure. Accordingly, there are many death cases due to infectious diseases, and according to the statistics of mortality on patients subjected to long term hemodialysis, 13.3% of death cases was due to infectious diseases. It is said that immunofunctional failure in renal failure appears as the cellular immunofunctional failure rather than the humoral one (refer to Clinical Immunology (Japan), 12(8), 607-614, 1980).
The infection in various renal diseases can be prevented by preventing or recovering the reduction of immunofunction.
After studying for finding the agent which prevents the reduction of immunofunction concerning renal diseases, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 is effective for that purpose, and have arrived at the present invention.
Since the cellular immunofunctional failure appears more remarkably than the humoral one in the renal diseases, the present inventors have examined mainly the effect of the present compound on the thymus which controls the cellular immunofunction. As the results of administration of the present compound to experimental animals, it is found that the present compound prevents the reduction of the following factors: the weight ratio of thymus to whole body, the number of thymocytes, the pattern of the size of thymic cells and blastoid-transformation of lymphocytes.
Namely, the present compound is useful as an agent for preventing the reduction of immunofunction in renal diseases for instance, chronic renal failure, uremia, and nephrotic syndrome.
In the next place, the activity of the present compound in controlling magnesium-metabolism will be explained as follows:
Magnesium stands fourth in cations within the body in abundance, and within cells magnesium stands next to potassium. Its abundance in extracellular fluid stands in the descending order of sodium, potassium, calcium and magnesium. Magnesium within the cells is in close connection to the activities of many enzymes, and magnesium in the extracellular fluid concerns the excitation of nerves and muscles. Moreover, magnesium is the essential element to the functions of a number of enzymes, for instance, those in glycolysis, and concentration of magnesium in serum is kept within a fixed range. One third of the magnesium in bone presents within the superficial layer of bone, particularly in the surface of hydroxyapatite to flow out of the osseous tissue for keeping the equilibrium with magnesium in serum.
The abnormality in magnesium metabolism has been observed in various morbid cases in spite of its equilibrium within the body (refer to Japanese J. Clin. Med., 38 (special spring number) 275-287, 1980).
Recently, studies on magnesium metabolism in the patients suffering from renal failure and receiving homodialysis have been carried out, and it is suggested that the serum level of magnesium is raised by actual hemodialysis and that there is a relationship between the serum level of magnesium and metastatic calcification.
Accordingly, it becomes possible to alleviate, prevent or improve various diseases by returning the abnormal magnesium metabolism to the normal one.
In consideration of the above-mentioned situation, development of an agent for normalizing the abnormalized magnesium metabolism has been demanded.
As a result of studying on the endogenous substances present within healthy human body, of which the safety has been confirmed, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 is able to control the metabolism of magnesium, and based on the finding, the present inventors have attained the present invention.
For reference, concerning the metabolism of magnesium, the following articles have been publicly reported:
(1) Proceeding of XVII KAWAGUCHI-KO Conference on the relationship between hormones and water or electrolytes, 155-167, 1981. Ed. ISIYAKU-Publishing Co. (Japan).
The present compound administered once to the rats subjected to thyroidectomy and parathyroidectomy did not affect the magnesium level in serum thereof and the magnesium clearance thereof.
(2) Pavlovitch, J. H., J. Clin, Invest., 68, 803-810 (1981).
The present compound preliminarily administered to the rats subjected to bilateral nephrectomy did not affect significantly the magnesium level in serum thereof.
The present inventors could not find any report which informs the beneficial effect of the present compound on the metabolism of magnesium.
It has been also known that there are no definite relationships between calcium and metabolism of magnesium (refer to Walsor, M. J., J. clin. Invest., 40, 723-730, 1961).
The agent for controlling metabolism of magnesium according to the present invention is the agent for restoring the abnormal state of concentration of magnesium within living body to the normal state and for preventing the abnormal change of the concentration of magnesium within living body.
The abnormality in magnesium metabolism has been observed in various diseases, for instance, renal diseases nerval diseases, diseases due to abnormal osseous metabolism, diseases on internal secretion, digestive troubles and diseases of circulatory system, and accordingly, the present compound makes it possible to alleviate, improve or prevent these diseases.
The anti-hyperphosphatemic activity of the present compound is described as follows:
Hyperphosphatemia has become an issue recently. It occurs mainly due to the reduction of excreting function (increase of reabsorption) in the kidneys in the morbid state such as that of chronic renal failure, hypoparathyroidism, acromegaly and acute disused-bone atrophy.
In the recent years, the number of patients receiving a long-term hemodialysis due to chronic renal failure goes on increasing and because their frequent development of a complication of symptoms such as metastatic calcification and metabolic acidosis due to their hyperphosphatemia, inorganic phosphate within their blood has been controlled by the increase in frequency of dialysis, the restriction of phosphorous in their diet and the administration of alumina gel as the binder of inorganic phosphate in their bodies. Although almost every patient receiving hemodialysis is treated by alumina gel-administration to reduce serum inorganic phosphate level, according to the recent studies, it has been elucidated that aluminum accumulates in the patient's brain due to the administered alumina gel and accordingly, the method of treatment by administration of alumina gel is extremely dangerous. As another method for treating hyperphosphatemia, administration of calcitonin has been tried. Although calcitonin's pharmacological function to reduce serum inorganic phosphate level is strong enough for 2 to 3 hours after its administration, since it is peptide hormone, persistency of its effectiveness is poor, and more over, since the state in the patient's body just after the administration of calcitonin differs drastically from the state when the calcitonin's effect has disappeared, the administration is apt to become a very hard treatment for the patient.
Still more, the administration of a peptide hormone derived from a foreign animal (for instance swine, eel and the like) gives an poorer effectiveness when continuously administered than the effectiveness when it was at the first time administered because of its antibody production, and there is a danger of anaphylaxis.
In addition, calcitonin's effectiveness can not be exhibited when orally administered, and accordingly, it is given by intramuscular injection and such a method of administration is accompanied by the patient's pain. Thus, it cannot be said that calcitonin is a safe pharmaceutic. In consideration of the above-mentioned fact, development of an agent safely usable for treating hyperphosphatemia has been demanded.
As a result of studying for finding a suitable compound among those substances which are endogenous within a healthy human body and high in safety, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 has an activity of reducing the inorganic phosphate level in serum, and based on the finding, they have attained the present invention.
It was found by the present inventors that administration of 24R,25-(OH).sub.2 -D.sub.3 to the rats in a state of hyperphosphatemia due to 5/6-nephrectomy (resulting in one third of one kidney remaining) caused the significant reduction of the inorganic phosphate level in the rat's serum. It was also found by the present inventors that administration of 24R,25-(OH).sub.2 -D.sub.3 to the Wistar rats to which puromycin-amino-nucleoside (produced by Sigma Co., hereinafter abbreviated and referred to as AN) had been administered to bring into the state of hyperphosphatemia caused the significant reduction of the inorganic phosphate level in their serum.
According to the above-mentioned findings, the present inventors administered 24R,25-(OH).sub.2 -D.sub.3 to a patient of chronic renal failure showing a symptom of hyperphosphatemia to find out that the inorganic phosphate level in the patient's serum was significantly reduced to the normal level of 2.5 to 5.5 mg/dl (concerning the normal level, refer to Wajima, T., "Clinical Chemical Analyses" Chapter V, Electrolytes, page 117, 1979, Ed. Tokyo Kagaku Dojin).
In addition, it was found by the present inventors that administration of 24R,25-(OH).sub.2 -D.sub.3 to a patient of chronic renal failure who has been given hemodialysis with a dialyzing liquid containing 9 to 10 mg of calcium component/ml caused a significant reduction of the inorganic phosphate level in his serum to the normal level. In this connection, since the normal calcium level in human serum is 8.8 to 10.4 mg/dl, the concentration of calcium component in the dialysate is preferably 9 to 10 mg/dl. However, since it has been elucidated that the hemodialysis carried out frequently on one patient over a long period of time while using a dialysate of a concentration of calcium component of 9 to 10 mg/dl causes osteomalacia, 1-.alpha.-25-(OH).sub.2 or 1-.alpha.-(OH)-D.sub.3 is used in recent years for preventing osteomalacia with a dialysate of a concentration of calcium component of 6 to 7.5 mg/dl. However, since the administration of 1-.alpha.-25-(OH).sub.2 -D.sub.3 or 1-.alpha.-(OH)-D.sub.3 promotes not only the resorption of calcium but also the resorption of phosphorous from the bone or the absorption of calcium and phosphorous from intestinal tract to raise the inorganic phosphate level in serum, the above-mentioned alumina gel is used in combination.
In this connection, the present inventors have found that the use of a dialysate of a concentration of calcium component of 9 to 10 mg/dl with the administration of the antihyperphosphatemic agent of the present invention reduces the inorganic phosphate level in serum and improves the osteomalacia without causing metastatic calcification.
The activity of the present compound in controlling blood sugar level will be described as follows:
Although various injections and internal medicines have been developed for controlling the hyperglycemic state due to glycosuria to the normal level, such injections and internal medicines have various difficult problems, and the development of safer and more effective agent for controlling blood sugar level has been eagerly demanded. As a symptomatic agent against hyperglycemia, agents containing insulin-derivative of a form of injection and agents containing sulfonylurea-derivative of a form suitable for internal administration are commercialized.
As the insulin composition, other than the regular insulin which exhibits its effect immediately after administration, however, of which the effect does not maintains for a long period, there is the depot insulin composition to which various proteins are admixed to make the absorption into the patient's body slow and zinc is further bonded to reduce the solubility to the humor.
The agents for treating glycosuria in a form of internal medicine have shown a progress from sulfonyl urea-derivative to biguanide-derivative, and new type of sulfonylurea-derivative has appeared recently.
In recent years, through the long term epidemiological studies, presence of a correlation between the disturbance of smaller blood vessel of the patient of glycosuria and the extent of blood sugar control has gradually been ensured. Namely, it is to be expected that the strict maintenance of the normal blood sugar level will possibly prevent the progress of disturbance of smaller blood vessels. In the existing treatment of glycosuria by insulin, the amount of insulin to be administered at a time and the number of times of administration of insulin are adjusted so as to maintain the blood sugar level at hunger, and the daily level of urine sugar as normal as possible. However, since insulin is a peptide hormone, its life span in a living body is short and its action is strong, the fluctuation of the blood sugar level within a day of the patient of glycosuria is apt to be different from that of a healthy person, and it is extremely difficult to maintain the level within a normal range. Moreover, even if insulin is orally administered, its pharmacological effect can not be expected.
In consideration of the above-mentioned facts, development of a safe and stabilized agent for controlling blood sugar has been eagerly awaited.
As a result of studying on the endogeneous substances present in human body, of which safety has been varified, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 has an activity to regulate the human blood sugar level, and based on the finding, they have arrived at the present invention.
Namely, while using rats on which urine sugar was positively confirmed as a result of intraperitoneal administration of streptozotocin, and reduction of the urine sugar level and blood sugar level was shown as a result of administration of regular insulin and which showed a high urine sugar level and high blood sugar level again after a few days of the suspension of insulin-administration as model animal of glycosuria, the present inventors examined the endogeneous substances present in human body for finding a substance having an activity of regulating the blood sugar level within the rat body, and they have found that 24,25-(OH).sub.2 -D.sub.3 is active in blood sugar level control. The agent for blood sugar controlling according to the present invention exhibits the activity to reduce the blood sugar level by the oral administration of only a few micrograms per kg body weight, and in this sense, it is of a new type never seen hitherto. Moreover, in the case where the agent of the present invention was administered to patients suffering from glycosuria, significant reduction of the blood sugar level was observed. Namely, the agent for blood sugar controlling according to the present invention can exhibit its blood sugar controlling action even in human case.
In the next place, anti-tumour activity of the present compound will be described as follows:
The anti-tumour agent actually in use at present includes alkylating agents, metabolic-antagonist, anti-biotics, plant alkaloids and immunotherapeutic agents and among them, many agents which show in vitro cytotoxic effect are also strong in their side effect.
As a result of examining the activity of the endogeneous substances present in living human body against tumour, the present inventors have found that 24,25-(OH).sub.2 -D.sub.3 shows in vivo as well as in vitro cytotoxic effect against various cancer cells and that even when it is administered over a long period it does not show any side effects.
Namely, in the present inventors' study for examining in vitro anti-tumor activity of 24,25-(OH).sub.2 -D.sub.3 against both K-562 cells derived from human leukemia and LICR-LON-HMy 2 cells derived from human myeloma, an action of inhibiting proliferation of these tumor cells or a cytotoxic activity against these tumor cells was recognized. Moreover, also in the experiments using mouse and rat as the host, anti-tumour effect of the compound was confirmed.
On the other hand, even after administering every day for 30 days 24,25-(OH).sub.2 -D.sub.3 in vivo at a daily dose rate of 1 mg/kg, any significant abnormal findings were not observed in the biochemical examination of the blood and the autopsy of the host animal. Moreover, in the observation of the appearance of the thus treated animals, the results of biochemical examination and the autopsy during and/or after 2 weeks of the single administration of 24,25-(OH).sub.2 -D.sub.3 at a dose rate of 150 mg/kg body weight, no abnormal findings were obtained. The present compound is an endogeneous substance and a safe anti-tumour agent of new type.
24,25-(OH).sub.2 -D.sub.3 has been also publicly known, for instance, it has been disclosed in Pharmacia (Japan), 10, 319-322, 1974. The chemical structural formulae of 24,25-(OH).sub.2 -D.sub.3 and its two optically isomeric molecules thereof are shown as follows: ##STR1##
24,25-(OH).sub.2 -D.sub.3 herein mentioned includes 24R,25-(OH).sub.2 -D.sub.3, 24S,25-(OH).sub.2 -D.sub.3 and their mixtures.
24R,25-(OH).sub.2 -D.sub.3 is particularly preferable.
The pharmaceutical composition according to the present invention contains the above-mentioned substance as the active ingredient, and is used in one of the various states mentioned below.
Although the pharmaceutical agent according to the present invention can be administered orally, parenterally or via rectal route, oral administration is preferable.
The pharmaceutical composition containing 24,25-(OH).sub.2 -D.sub.3 of the present invention as the active ingredient is used in one of the following states for administration, namely, tablet, powder, granule, suppository, capsule, alcoholic solution, such as ethyl alcohol, isopropyl alcohol, ethylene glycol and propylene glycol solution, oil solution, aqueous suspension, etc. As a solvent for oil solution, triglyceride ester of fatty acid such as capric acid and caprylic acid, corn oil, cotton seed oil, peanut oil, fish liver oil, oily esters, and the like are used. In addition, cacao butter and glycerol are desirable. Furthermore, as other components of the pharmaceutical composition, lactose, starch, talc, magnesium stearate, sorbic acid, sorbate salts, saccharides or their alcoholic derivatives, physiological saline solution, surfactants, antioxydants and the like are used in combination with the present compound.
The content of the present substance in the pharmaceutical composition is 2.times.10.sup.-8 to 4.times.10.sup.-1 % by weight, preferably 2.times.10.sup.-5 to 1.times.10.sup.-1 % by weight, the daily dose of the present compound to an adult patient (of average body weight of 50 to 60 kg) being 1.times.10.sup.-2 to 1.times.10.sup.5 micrograms, preferably 5.times.10.sup.-1 to 1.times.10.sup.4 micrograms.
The result of acute toxicological test of the present compound is as follows: